Sanglard D, Ischer F, Koymans L, Bille J
Institut de Microbiologie, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.
Antimicrob Agents Chemother. 1998 Feb;42(2):241-53. doi: 10.1128/AAC.42.2.241.
The cytochrome P-450 lanosterol 14alpha-demethylase (CYP51A1) of yeasts is involved in an important step in the biosynthesis of ergosterol. Since CYP51A1 is the target of azole antifungal agents, this enzyme is potentially prone to alterations leading to resistance to these agents. Among them, a decrease in the affinity of CYP51A1 for these agents is possible. We showed in a group of Candida albicans isolates from AIDS patients that multidrug efflux transporters were playing an important role in the resistance of C. albicans to azole antifungal agents, but without excluding the involvement of other factors (D. Sanglard, K. Kuchler, F. Ischer, J.-L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378-2386, 1995). We therefore analyzed in closer detail changes in the affinity of CYP51A1 for azole antifungal agents. A strategy consisting of functional expression in Saccharomyces cerevisiae of the C. albicans CYP51A1 genes of sequential clinical isolates from patients was designed. This selection, which was coupled with a test of susceptibility to the azole derivatives fluconazole, ketoconazole, and itraconazole, enabled the detection of mutations in different cloned CYP51A1 genes, whose products are potentially affected in their affinity for azole derivatives. This selection enabled the detection of five different mutations in the cloned CYP51A1 genes which correlated with the occurrence of azole resistance in clinical C. albicans isolates. These mutations were as follows: replacement of the glycine at position 129 with alanine (G129A), Y132H, S405F, G464S, and R467K. While the S405F mutation was found as a single amino acid substitution in a CYP51A1 gene from an azole-resistant yeast, other mutations were found simultaneously in individual CYP51A1 genes, i.e., R467K with G464S, S405F with Y132H, G129A with G464S, and R467K with G464S and Y132H. Site-directed mutagenesis of a wild-type CYP51A1 gene was performed to estimate the effect of each of these mutations on resistance to azole derivatives. Each single mutation, with the exception of G129A, had a measurable effect on the affinity of the target enzyme for specific azole derivatives. We speculate that these specific mutations could combine with the effect of multidrug efflux transporters in the clinical isolates and contribute to different patterns and stepwise increases in resistance to azole derivatives.
酵母细胞色素P-450羊毛甾醇14α-去甲基酶（CYP51A1）参与麦角甾醇生物合成中的一个重要步骤。由于CYP51A1是唑类抗真菌剂的作用靶点，该酶可能易于发生改变从而导致对这些药物产生耐药性。其中，CYP51A1对这些药物的亲和力降低是有可能的。我们在一组来自艾滋病患者的白色念珠菌分离株中发现，多药外排转运蛋白在白色念珠菌对唑类抗真菌剂的耐药性中起重要作用，但并不排除其他因素的参与（D. Sanglard、K. Kuchler、F. Ischer、J.-L. Pagani、M. Monod和J. Bille，《抗菌药物化疗》39:2378 - 2386，1995年）。因此，我们更详细地分析了CYP51A1对唑类抗真菌剂亲和力的变化。设计了一种策略，即对来自患者的连续临床分离株的白色念珠菌CYP51A1基因在酿酒酵母中进行功能表达。这种筛选方法与对唑类衍生物氟康唑、酮康唑和伊曲康唑的药敏试验相结合，能够检测不同克隆的CYP51A1基因中的突变，这些基因的产物对唑类衍生物的亲和力可能受到影响。这种筛选方法能够检测到克隆的CYP51A1基因中的五种不同突变，这些突变与临床白色念珠菌分离株中唑类耐药性的出现相关。这些突变如下：第129位的甘氨酸被丙氨酸取代（G129A）、Y132H、S405F、G464S和R467K。虽然在一株唑类耐药酵母的CYP51A1基因中发现S405F突变是单个氨基酸取代，但在单个CYP51A1基因中同时发现了其他突变，即R467K与G464S、S405F与Y132H、G129A与G464S以及R467K与G464S和Y132H。对野生型CYP51A1基因进行定点诱变，以评估这些突变对唑类衍生物耐药性的影响。除G129A外，每个单一突变对靶酶对特定唑类衍生物的亲和力都有可测量的影响。我们推测，这些特定突变可能与临床分离株中多药外排转运蛋白的作用相结合，并导致对唑类衍生物耐药性的不同模式和逐步增加。
